Brendon Conlan, Rosemary Birch, Celine Kelso, Sophie Holland, Amanda P. De Souza, Stephen P. Long, Jennifer L. Beck, and Spencer M. Whitney

The folding and assembly of Rubisco large and small subunits into L₈S₈holoenzyme in chloroplasts involves many auxiliary factors, including the chaperone BSD2. Here we identify apparent intermediary Rubisco‐BSD2 assembly complexes in the model C₃‐plant tobacco. We show BSD2 and Rubisco content decrease in tandem with leaf age with approximately half of the BSD2 in young leaves (~70 nmol BSD2 protomer.m²) stably integrated in putative intermediary Rubisco complexes that account for <0.2% of the L₈S₈pool. RNAi‐silencing BSD2 production in transplastomic tobacco producing bacterial L₂ Rubisco had no effect on leaf photosynthesis, cell ultrastructure or plant growth. Genetic crossing the same RNAi‐bsd2 alleles into wild‐type tobacco however impaired L₈S₈ Rubisco production and plant growth, indicating the only critical function of BSD2 is in Rubisco biogenesis. Agrobacterium mediated transient expression of tobacco, Arabidopsis or maize BSD2 re‐instated Rubisco biogenesis in BSD2‐silenced tobacco. Overexpressing BSD2 in tobacco chloroplasts however did not alter Rubisco content, activation status, leaf photosynthesis rate or plant growth in the field or in the glasshouse at 20°C or 35°C. Our findings indicate BSD2 functions exclusively in Rubisco biogenesis, can efficiently facilitate heterologous plant Rubisco assembly and is produced in amounts non‐limiting to tobacco growth.